1. Field of the Invention
This invention relates to the inactivation of enzymes, more particularly to a method of reducing enzyme activity in blood serum, e.g. human blood serum.
2. Prior Art
Blood serum is a complex biological fluid containing numerous components of substantial physiological importance. In a normal healthy person, the concentrations of these components fall within certain reasonably well defined limits. When one or more of these components is determined upon analysis to fall outside of these acceptable limits, various diseases or pathological conditions of the body system are indicated. The rapid analysis of various blood serum components has thus became a valuable adjunct in the clinical diagnosis of disease. In recent years, novel automated and non-automated procedures and assay kits have been developed for the rapid analysis of the multiple components of blood serum. These procedures and kits are capable of determining the concentrations of a host of blood serum enzyme levels in a blood sample, for example, alkaline phosphatase, lactate dehydrogenase (LDH), transaminases (SGOT), and (SGPT), (GGT), and creatinine phosphokinase (CPK). Immunoassay procedures are available for determining the serum levels of components such as gastrin and insulin.
In the performance of such analytical tests on blood serum and other biological samples, it is necessary to use laboratory serum standards for purposes of calibration and control testing. Accurate results in the use of these test procedures and kits are dependent upon such serum standards.
Such standards for calibration and control are often difficult, if not impossible to obtain by known procedures. For example, in the determination of certain specific enzymes, e.g. alkaline phosphatase, GGT, LDH, lipase, SGOT, it is difficult and expensive to obtain stable standard serums, particularly human serum having a controlled or reduced, e.g. zero, level of such enzymes. For example, lipase free injectable human serum albumin must often be used as a substitute for lipase free human serum and bovine serum albumin is often used as a standard or base matrix for calibrators.
Enzyme deactivation is a known process and may be accomplished, for example, by heating the enzyme to a sufficient temperature to inactivate the enzyme. Such a method of inactivating enzymes is impractical when seeking to inactivate enzymes in serums, particularly human serums, for such heat treatment will generally destroy the usefulness of the serum and its components.
Alternatively, a number of chemical enzyme inactivators have been developed. Representative of such chemical inactivators are copper sulfate, barium peroxide, hydrogen peroxide, silver nitrate, mercuric chloride, sodium hypochloride, and cadmium chloride. Most of these inactivators have not been used to treat serums, however, if used, they would tend to be non-selective in that they would destroy the usefulness of the serum by destroying certain components thereof. Additionally, a number of these chemical inactivators are toxic, would discolor the serum and are relatively expensive to use.
More specifically, U.S. Pat. No. 2,647,854 to Pfannmuller et al describes the inactivation of enzymes with barium peroxide. The enzymes, e.g. amylases, are used to thin water suspensions of raw starch in preparing paper and textile sizes, starch coatings, laminating adhesives and the like. Acetic acid may be used to decrease the pH of the solution to thereby increase the solubility of the barium peroxide. Chemical inactivation with barium peroxide may also be utilized on other enzyme types.
U.S. Pat. No. 3,513,072 to Frankevicz et al is directed to the inactivation of alpha-amylase enzymes by fluorosilica compounds, to control starch liquefaction in a starch slurry.
U.S. Pat. No. 3,806,421 to Ueda et al describes an inhibitor for retarding or preventing decomposition of starch to glucose by amylase. The inhibitor is recovered from the culture broth of Streptomyces flavochromogenes 280.
U.S. Pat. No. 4,007,008 to Becker et al describes a method of treating animal serum to simulate human blood serum for use as a reference standard for automated biological testing instruments. Non-human serum, e.g. serum of bovine, equine, porcine, sheep, etc. are treated to decrease the enzyme activities in such serum to human levels, without alteration of other constituent levels normally found in human blood serum. Becker et al accomplishes such by elevation of the pH of the serum with a base, followed by neutralization with an acid to a normal pH. Of additional relevance is Example 2 in Becker et al which describes decolorizing natural serum by the controlled addition of small amounts of a mild oxidant.
U.S. Pat. No. 4,086,139 to Hoerle describes freeing the protease enzyme in a mixed protease-amylase enzyme composition of its amylase activity by treating the composition with " . . . an oxidizing agent selected from the group consisting of chlorite and hypo-chlorite ions . . . " The ions are added to the mixture in an amount sufficient to inactivate the amylase to a greater degree than the protease. A preferred hypochlorite composition is CLOROX.
U.S. Pat. No. 4,184,848 to Batz et al describes the use of a specific group of polyethylene glycol esters for the elimination of turbidity in serum.
U.S. Pat. No. 4,264,471 to Briggs describes a process for obtaining delipified human serum or plasma of low turbidity. The process comprises simultaneously deionizing and pH adjusting the serum of plasma to near the isoelectric point of the lipoprotein.
Additionally, Sprossig et al, "Kaltsterilisation Von Seren Mit Peressigsaure", Journal of Hygiene, Epidemiology, Microbiology and Immunology, 20, 1976, No. 2 157-163 describes the use of low concentrations, i.e. 0.02% to 0.1%, of peracetic acid to sterilize serum used to prepare a culture medium for bacteria, mycoplasmas and tissue cultures.
Of additional interest are U.S. Pat. Nos. 3,986,930 to Kurooka et al and 4,279,994 to Huang which describe the determination of lipase enzyme in serum using chemical reagents containing sulphur. These patents also teach the use of enzyme inhibitors which specifically inactivate enzymes which interfere with the determination of lipase without inactivating lipase. These enzyme inhibitors are exemplified by phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate.
None of the aforementioned references, teach or suggest the invention described and claimed herein or the advantages in utilizing such invention, particularly in the treatment of human serum to produce a standard or control serum.